The CD8 cell surface glycoprotein is an important signaling molecule functioning as a coreceptor with the T cell receptor (TCR) for interaction with MHC class I. CD8 is a dimer of an alpha and beta polypeptide or a homodimer. The focus of this grant is on the CD8b chain. Changes in sialylation on O-linked sugars of the CD8b chain during T cell development modulate CD8 interaction with MHC class I. We have three aims (i) Determine the role of CD8b in CD8ab-MHC interaction. We will create a panel of human and murine CD8b mutants and determine the effect on interaction with MHC class I tetramers. We will make monoclonal antibodies (mAbs) to the human CD8b protein by immunizing CD8B KO animals with mouse cells expressing the human CD8b protein. These antibodies as well as murine antibodies will be tested for blocking or enhancing CD8 interaction with MHC class I tetramers and the mAb epitopes mapped with the panel of CD8b mutants. The location of epitopes for blocking, enhancing or mAbs with no effect will enhance our understanding of CD8-MHC class I interaction; (ii) Determine the functional consequence of human CD8b isoforms with different cytoplasmic tails. We will determine the expression pattern of the different isoforms in thymocytes, and mature T cells that are either resting, activated, or memory T cells. We will express each isoform in a human T cell line that expresses CD8a but not CD8b and determine the effect on raft localization and signal transduction; (iii) Examine the role of the CD8b chain in promoting clustering of CD8 molecules. Multimers of CD8 exist on thymocytes. We will determine if CD8b is required to form these multimers using CD8B KO animals. To study the role of glycosylation on clustering of CD8 molecules, we will express CD8aa or ab in a mutant chinese hampster ovary line (CHO) which cannot add O-linked sugars to glycoproteins unless exogenous sugars are added to the medium. We will analyze the binding of MHC class I tetramers to the different glycosylated forms of CD8 and analyze CD8 biochemically with or without crosslinking agents. Because the affinity of a T cell clone is regulated by the proportion of CD8ab vs CD8aa and by changes in sialylation of the CD8b chain, this protein is an important determinant for whether a T cell becomes activated. If the human CD8b isoforms are functionally distinct and the expression pattern between people is different, this could influence susceptibility to infection or autoimmunity. Understanding how CD8 binds to MHC class I tetramers and mapping enhancing or blocking antibodies will Contribute to this new technology.